Tacrolimus (TAC) is a widely prescribed immunosuppressive agent for solid organ transplantation to prevent allograft loss. It inhibits activation and proliferation of CD4+ and CD8+T-lymphocytes by binding to immunophilin FK506-binding protein (FKBP12) as well as inhibiting calcineurin phosphatase and subsequent IL-2 production. TAC is highly lipophilic and is excreted from the body following metabolism by the CYP450 3A4/5 enzymes. Due to genetic polymorphisms in the drug metabolizing enzyme CYP3A, bioavailability of TAC can vary significantly among individuals. In addition, TAC is a substrate for the P-glycoprotein efflux transporter (Pgp), also known as multidrug resistance protein 1 (MDR1), a cell membrane ATP-dependent efflux pump encoded by the ABCB1 gene with broad substrate specificity. Differences in the expression level of MDR1 and ABCB1 may also contribute to inter-individual variability of TAC bioavailability. As a result of its narrow therapeutic index and high variability in bioavailability, ongoing monitoring of TAC is essential for maintaining optimal therapeutic concentrations to ensure allograft survival and reduce nephrotoxicity.
TAC concentrations in peripheral blood mononuclear cells (PBMCs) and biopsy from implanted tissues have shown to be good indicators for therapeutic efficacy and predictors for allograft rejection in liver and kidney transplant recipients. Because of the invasiveness of biopsy and complicated sample preparation procedures, the use of these specimens in routine TDM is not practical. TAC blood levels have been shown to have poor correlation with, in situ levels, in lymphocytes and tissues as well as with unbound fractions. As a result, whole blood sampling fails to provide a reliable prediction of allograft status and toxicity. Accordingly, there is a need in the art for a less invasive method that provides reliable prediction of allograft status and total exposure to free/pharmacologically active form of TAC.